PROJECT SUMMARY/ABSTRACT For COVID-19, persons who inject drugs (PWID) have been identified as a population at high-risk of exposure to SARS-CoV-2 (the virus that causes COVID-19) because of their increased risk of homelessness or incarceration?situations linked to increased rates of the disease transmission and co-infection with HIV-1. Given that a SARS-CoV-2 vaccine is likely 12?24 months away, there is a critical unmet medical need for medical countermeasures that could contain COVID-19 outbreaks in the general population and in these difficult-to-reach high-risk populations such as PWIDs in particular. Moreover, there is a fundamental gap in our understanding of SARS-CoV-2 infection and pathogenesis in these at-risk PWID populations. Evidence indicates that SARS- CoV-2 infects and depletes T lymphocytes and many HIV-infected PWID have limited access to antiretroviral therapy and consequently exhibit pre-existing CD4+ T-cell depletion. Hence, SARS-CoV-2 infection could accelerate clinical progression to AIDS, or alternatively, SARS-CoV-2 infection in HIV+ PWID could exacerbate COVID-19 clinical symptoms leading to elevated risk of death. Thus, HIV+ PWID may be at elevated risk of death from SARS-CoV-2 infection. In these PWID populations, reducing T-cell depletion would be highly beneficial to halting clinical progression and may a viable long-term therapeutic goal. The specific objective of this supplement proposal is to repurpose existing technologies to rapidly develop a Gene Drive Therapy (GDT) candidate for SARS-CoV-2 and quantify its breadth of interference and transmission in vitro in patient T-cells from HIV+ PWID. This effort will build heavily off our recent success in engineering an HIV-1 GDT (see Parent Award) and a GDT against Zika Virus (ZIKV), demonstrating that the GDT concept can be repurposed for other viruses. The central hypothesis?based on extensive preliminary studies in HIV and ZIKV?is that a putative SARS-CoV-2 GDT, depleted of all the pathogenic viral genes, could target the same cells as wild-type SARS CoV-2 (including T lymphocytes), compete for intracellular resources, and reduce SARS CoV-2 viral load and pathogenesis, thereby serving as a single-administration therapeutic. The rationale for a GDT countermeasure for SARS CoV-2 is based on extensive data for HIV-1 in humanized mice and positive FDA meetings. We will achieve our objectives via two specific aims: (i) Engineer a SARS-CoV-2 GDT candidate (by adapting the existing Bioreactor platform); and (ii) Test the SARS CoV-2 GDT candidate's protective effect on patient T-cells from an HIV+ PWID cohort in Tijuana Mexico. While the GDT approach carries inherent risks, single-administration therapeutics would be highly beneficial particularly for treating difficult-to-reach, high-risk PWID populations. Regardless of the success of GDTs in protecting against T-cell depletion, the studies proposed here will have broad fundamental significance by assaying how SARS-CoV-2 infection impacts T lymphocytes from HIV+ PWID. These studies would also provide validation of a novel medical countermeasure with the potential to be rapidly deployed against new viral threats.